An effective and safe gene delivery system is required for gene therapy to be clinically useful. Viral vectors are relatively efficient gene delivery systems, but suffer from a variety of limitations, such as the potential for reversion to the wild type as well as immune response concerns. As a result, nonviral gene delivery systems are receiving increasing attention (Worgall, et al., Human Gene Therapy 8:37-44 (1997); Peeters, et al., Human Gene Therapy 7:1693-1699 (1996); Yei, et al., Gene Therapy 1:192-200 (1994); Hope, et al., Molecular Membrane Biology 15:1-14 (1998)). Plasmid DNA-cationic liposome complexes are currently the most commonly employed nonviral gene delivery vehicles (Felgner, Scientific American 276:102-106 (1997); Chonn, et al., Current Opinion in Biotechnology 6:698-708 (1995)). However, complexes are large, poorly defined systems that are not suited for systemic applications and can elicit considerable toxic side effects (Harrison, et al., Biotechniques 19:816-823 (1995); Huang, et al., Nature Biotechnology 15:620-621 (1997); Templeton, et al., Nature Biotechnology 15:647-652 (1997); Hofland, et al., Pharmaceutical Research 14:742-749 (1997)).
Recent work has shown that plasmid DNA can be encapsulated in small (˜70 nm diameter) “stabilized plasmid-lipid particles” (SPLP) that consist of a single plasmid encapsulated within a bilayer lipid vesicle (Wheeler, et al., Gene Therapy 6:271-281 (1999)). These SPLPs typically contain the “fusogenic” lipid dioleoylphosphatidyl-ethanolamine (DOPE), low levels of cationic lipid, and are stabilized in aqueous media by the presence of a poly(ethylene glycol) (PEG) coating. SPLP have systemic application as they exhibit extended circulation lifetimes following intravenous (i.v.) injection, accumulate preferentially at distal tumor sites due to the enhanced vascular permeability in such regions, and can mediate transgene expression at these tumor sites. The levels of transgene expression observed at the tumor site following i.v. injection of SPLP containing the luciferase marker gene are superior to the levels that can be achieved employing plasmid DNA-cationic liposome complexes (lipoplexes) or naked DNA. Still, improved levels of expression may be required for optimal therapeutic benefit in some applications (see, e.g., Monck, et al., J. Drug Targ. 7:439-452 (2000)).
Typically, both liposomes and SPLPs comprise cationic lipids. Often the cationic lipids have 2 alkyl chains, ether (oxygen) bonds, and an amine head group such as, e.g., DODAC and DODMA. Typically the alkyl chains comprise a single site of unsaturation. Unfortunately, cationic lipids with only a single site of unsaturation can lack flexibility. Liposomes or SPLP comprising these cationic lipids can lack sufficient membrane fluidity, thus impacting the efficiency of delivery of a bioactive agent to a cell or to a patient.
Lipid flexibility is important in the development of liposomal or SPLP drug delivery systems. Therefore, it is desirable to develop cationic lipids that are more flexible, thereby, increasing the membrane fluidity of the liposomes or the SPLP. The present invention addresses this and other needs.